Aim of the study To investigate the effects of P27RF-Rho on

Aim of the study To investigate the effects of P27RF-Rho on hepatocellular carcinoma (HCC) cell growth and explore the possibility of using it as a novel therapeutic target for liver cancer treatment. growth, and P27RF-Rho is probably a promising target for HCC treatment. [4]. Deletion or downregulated expression of DLC-1 is common in HCC. Restoration of DLC-1 expression in HCC cells resulted in caspase-3-mediated apoptosis, inhibition of cell growth, and invasiveness DH5a was purchased from Invitrogen. Restriction enzymes and DNA ligase were from Thermo Fisher Scientific Company (USA). DNA gel recovery kit and kit for rapid extraction of plasmid DNA were purchased from Promega Corporation (USA). Kits for quantitative PCR and RT-PCR, as well as DNA Marker DL2000, were purchased from Takara Company (China). Lipofectamine 2000 and Annexin V-FITC apoptosis detection kit were from Invitrogen (USA). Real-time PCR System ZD4054 (Stratagene Mx3005p) was purchased from Agilent Technologies Co. Ltd (USA). Flow Cytometry was purchased from BD FACSCalibur (BD, USA). All antibodies were purchased from Santa Cruz (USA). All enzymes were purchased from Takara. Construction of shRNA vectors Two shRNA vectors, one for P27RF-Rho knockdown and another for negative control, were designed and constructed. The RNA interference sequence targeting P27RF-Rho was 5 – GGAGCTGGTTGTACAGTTTTCAAGAGAAACTGTACAACCAGCTCC TTTTT – 3(sense), and 5 – AAAAAGGAGCTGGTTGTACAGTTTCTCTTGAAAACTGTACAACCAGCTCC GTAC – 3(antisense). Negative control (scramble) was 5 – GTTGCATACGTGCGGTGATAT TCAAGAGATATCACCGCACGTATGCAAC TTTTT ZD4054 – 3(sense) and 5 – AAAAAGTTGCATACGTGCGGTGATATCTCTTGAATATCACCGCACGTATGCAAC GTAC – 3(antisense). The shRNA vector was purchased from Huijun Biotechnology Company (China), and the oligo DNA fragments were synthesised by this company. Double-stranded DNA was obtained by oligo DNA annealing. In brief, sense and antisense oligonucleotides (100 pmol) 2 l were mixed with 10 annealing buffer 2 l and ddH2O 14 l in an Eppendorf tube, with a total reaction volume of 20 l. The annealing procedure began at 94C for three minutes, then the temperature was lowered with a gradient of 5C, and kept at each temperature gradient for 5 minutes, until room temperature was reached. After enzyme digestion with Kpn and HPaI. ShRNA vector and the double stranded DNA were ligated with T4 DNA ligase. The ligation mixture was incubated at 16C overnight. It was transformed into DH5a. After transformation, positive clones were identified by PCR and sequenced. ZD4054 The two shRNA vectors were named U6-shRNA-CMV-copGFP-PGK-Puro-P27RF-Rho (P27RF-Rho-siRNA for short) and U6-shRNA-CMV-copGFP-PGK-Puro-Scramble (P27RF-Rho-Scramble for short). Lentivirus packaging and cell infection 1.2 107 HEK 293T cells were inoculated in a plate 60 mm in diameter, cultivated at 37C in a humidified incubator containing 5% CO2, and left to grow overnight. The cells were transfected with a mixture of shRNA vector 4.0 g, psPAX2 vector 2.0 g, and pMD2G vector 2.0 g, using Lipofectamine 2000 according to the manufacturers instructions. Transfection efficiency was determined by fluorescence microscope. Briefly, three non-overlapping vision fields were randomly selected, the number of positive cells out of every 100 cells in total was determined, and the corresponding transfection efficiency was then calculated according to the following formula: transfection efficiency = total number of positive cells in THREE vision fields/300 100%. The ZD4054 transfection efficiency above 90% was considered as a successful transfection. Supernatant containing lentivirus was harvested 48 and 72 hours after transfection, respectively, using Lenti-Pac? Lentivirus Concentration Solution (GeneCopoeia, USA), according to the manufacturers instructions. Cells were infected with the lentiviruses at Multiplicity of Infection (MOI) APRF of 20. Forty-eight hours after lentivirus infection, puromycin at the concentration of 5 g/ml was applied to screen stable infected cell lines. Transfection efficiency exceeded 95% in the screened cell lines. RT-PCR detection of P27RF-Rho silencing PP27RF-Rho knockdown was verified by RT-PCR. Briefly, total RNA was extracted according ZD4054 to TRIzol kit (Tiangen, China) instruction, and cDNA was synthesised from the extracted total RNA using.